Objective To analyze the differentially expressed genes in the transcriptome of Mycobacterium smegmatis mc²155 under the action of ethionamide (ETH) and explore the genes associated with the mechanism of action of ETH.

Methods The minimum inhibitory concentration (MIC) of ETH to mc²155 strain was determined using microplate broth dilution method. The mc²155 strain treated with 1/4 MIC ETH was used as the drug-treated group, while the strain without ETH treatment was used as control group. Total RNA was extracted for transcriptome sequencing analysis to identify differentially expressed genes (DEGs). Additionally, gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis were conducted for the DEGs. Nine DEGs were randomly selected and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to validate the transcriptome sequencing results.

Results Transcriptome analysis showed that there were 527 DEGs in the ETH-treated group compared with the control group, of which 174 genes were up-regulated and 353 genes were down-regulated. GO enrichment analysis showed that DEGs were mainly involved in molecular functions such as adenosine triphosphatase activity, hydrolase activity, transmembrane transporter activity, and biological processes such as transmembrane transport and localization. KEGG enrichment analysis results showed that DEGs were mainly concentrated in pathways such as biosynthesis of secondary metabolites, ATP-binding cassette transporters, microbial metabolism in different environments, and two-component systems. The RT-qPCR results of the DEGs were consistent with the trend of differential expression changes observed by transcriptome sequencing.

Conclusion Ethionamide can significantly affect the expression of Mycobacterium smegmatis genes, involving multiple functional genes and metabolic pathways. Further investigation is required to elucidate its specific regulatory mechanisms.